CN107815491B - Kit for rapidly and specifically detecting mycobacterium tuberculosis and use method thereof - Google Patents
Kit for rapidly and specifically detecting mycobacterium tuberculosis and use method thereof Download PDFInfo
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- CN107815491B CN107815491B CN201711167560.6A CN201711167560A CN107815491B CN 107815491 B CN107815491 B CN 107815491B CN 201711167560 A CN201711167560 A CN 201711167560A CN 107815491 B CN107815491 B CN 107815491B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention relates to a kit for rapidly and specifically detecting mycobacterium tuberculosis and a use method thereof, the kit comprises a working standard substance, a constant-temperature amplification reaction solution and a negative reference substance, the working standard substance comprises a positive reference plasmid of a specific conserved sequence of the mycobacterium tuberculosis, and the specific conserved sequence is SEQ ID No. 1; the isothermal amplification reaction solution comprises 3 pairs of primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; a loop primer LF with a sequence of SEQ ID No. 7; the negative control is sputum DNA extract of normal person. The kit can rapidly and specifically detect the mycobacterium tuberculosis, and has very good sensitivity and specificity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for rapidly and specifically detecting mycobacterium tuberculosis and a using method thereof.
Background
Mycobacterium tuberculosis (tuberculosis) is a pathogenic bacterium causing tuberculosis, can invade all organs of the body, but is most common in tuberculosis. Tuberculosis remains an important infectious disease to date. It is reported by WHO that about 800 new cases occur each year, and at least 300 million people die from the disease. The death rate of 300 people per 10 million before China is built, the death rate of people living in various diseases is the first, the living level of people is improved after China is built, the sanitary state is improved, group prevention and mass control are particularly developed, children are generally inoculated with BCG, and the morbidity and the mortality of tuberculosis are greatly reduced. However, it should be noted that some parts of the world have an increasing incidence due to aids, drug abuse, application of immunosuppressive agents, alcohol abuse, poverty and the like. Therefore, the timely, efficient and specific detection of the mycobacterium tuberculosis becomes an effective means for preventing and treating the diseases caused by the mycobacterium tuberculosis.
Bacterial culture and smear examination are one of the most widely used etiological diagnostic techniques, with sputum culture accounting for over 50%, but clinical compliance of traditional routine sputum specimen bacteriological tests is not high due to nonstandard sputum specimen collection and the complexity of microbial and human correlation.
The detection reagent for the mycobacterium tuberculosis at home and abroad is mainly based on antibody detection, nucleic acid detection, drug-sensitive medium detection and the like. At present, nucleic acid detection has become the main method for detecting mycobacterium tuberculosis; the detection of nucleic acids against mycobacterium tuberculosis is mainly of the following types: PCR-fluorescent probe method, fluorescent PCR melting curve method, PCR-reverse dot hybridization method, DNA microarray chip method, etc. However, the antibody detection sensitivity is low, the false positive phenomenon is more, the nucleic acid detection needs large-scale instruments such as a fluorescence PCR instrument, the nucleic acid extraction needs to be carried out on pathogenic bacteria in advance, the subsequent fluorescence detection consumes longer time, and the like.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, which utilizes DNA polymerase with strand displacement activity and designs two or three pairs of special primers aiming at 6 independent regions of DNA, and the reaction can be completed within 20-30 minutes under the reaction condition of 60-65 ℃. LAMP has the characteristics of rapidness, simplicity and strong specificity, and can hopefully replace the traditional PCR method to detect microorganisms. Therefore, the kit is invented aiming at a section of specific primer sequence of the mycobacterium tuberculosis, and the kit can rapidly and specifically detect the mycobacterium tuberculosis.
Disclosure of Invention
The invention provides a kit for rapidly and specifically detecting mycobacterium tuberculosis and a using method thereof, which can rapidly and accurately detect the mycobacterium tuberculosis in samples such as sputum, swabs and the like of patients in real time.
The technical scheme for solving the technical problems is as follows: a kit for rapidly and specifically detecting mycobacterium tuberculosis, which comprises a working standard substance, a constant-temperature amplification reaction solution and a negative reference substance,
the working standard comprises a positive control plasmid of a specific conserved sequence of the mycobacterium tuberculosis, and the specific conserved sequence is SEQ ID No. 1;
the isothermal amplification reaction solution comprises 3 pairs of primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; a loop primer LF with a sequence of SEQ ID No. 7;
the negative control substance is sputum DNA extracting solution of normal people.
On the basis of the above technical solutions, the present invention may further specifically select as follows.
Specifically, the positive control plasmid is constructed by cloning a synthetic fragment corresponding to a specific conserved sequence into a pGEM-T vector, and the concentration of the synthetic fragment is 104copies/μL。
Specifically, the solvent of the isothermal amplification reaction solution is double distilled water, and the isothermal amplification reaction solution contains solutes with the following concentrations: bst DNA polymerase 8U/20. mu.L; 8mM MgSO4；2.4mM each dNTPs；20mM KCl；20mM(NH4)2SO4(ii) a 40Mm Tris; 40Mm HCl; 0.2 wt% TritonX-100; 5 pmol/20. mu.L of outer primer F3; 5 pmol/20. mu.L of outer primer B3; 40pmol of inner primer FIP; 40pmol of inner primer BIP; 20pmol of loop primer LB; 20pmol of loop primer LB; 1.25 × EvaGreen fluorescent dye.
In addition, the invention also provides a method for using the kit for rapidly and specifically detecting mycobacterium tuberculosis, which comprises the following steps:
s1, preparing a sputum DNA specimen to be detected;
s2, taking three reaction tubes, adding 20 mu L of constant-temperature amplification reaction liquid into each reaction tube, then adding 5 mu L of sputum DNA specimen to be detected into the first reaction tube, adding 5 mu L of working standard substance into the second reaction tube, adding 5 mu L of negative reference substance into the third reaction tube, and then placing the three reaction tubes under constant temperature condition for amplification reaction;
and S3, after the amplification reaction is finished, reading fluorescence values of reaction liquid in the three reaction reverse tubes by using an enzyme-labeling instrument and the like, and comparing to determine whether the sputum DNA sample to be detected contains mycobacterium tuberculosis. And (3) measuring the fluorescence value in real time in the amplification reaction process, and determining whether the sputum DNA specimen to be detected contains the mycobacterium tuberculosis according to the amplification curve.
Specifically, the reaction temperature of the amplification reaction was 65 ℃ and the reaction time was 60 min.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, a section of specific conserved sequence containing 300bp is screened out by carrying out comparison analysis on a gene sequence of the mycobacterium tuberculosis, then 3 pairs of specific primers are designed aiming at the specific conserved sequence, the 3 pairs of specific primers, Bst DNA polymerase and EvaGreen fluorescent dye (the turbidity can bring errors for detection through naked eye observation, the detection sensitivity is not high, so that the fluorescent dye, necessary inorganic salt and buffer solution are added to form a constant-temperature amplification reaction solution, the DNA of the mycobacterium tuberculosis can be rapidly amplified in the constant-temperature amplification reaction solution, and the fluorescence real-time monitoring is carried out, so that the rapid detection of the mycobacterium tuberculosis is realized, and the method can be applied to the rapid detection of the mycobacterium tuberculosis in sputum or nasopharyngeal swab samples, and is convenient to operate and convenient and rapid to detect; when the kit is used, 5 mu L of each of the DNA of a sample to be detected, the working standard substance and the negative reference substance is respectively added into three reaction tubes filled with 20 mu L of the constant-temperature amplification reaction solution, and the fluorescence values monitored in real time are compared in the amplification reaction process, so that the detection can be rapidly finished.
The kit provided by the invention has very good sensitivity and specificity: the kit is matched with fluorescence detection equipment, the lowest detection limit is 10 copies/mu L, DNA is extracted from common lung pathogenic bacteria such as pseudomonas aeruginosa, streptococcus pneumoniae, haemophilus influenzae, legionella pneumophila, mycoplasma pneumoniae, klebsiella pneumoniae, staphylococcus aureus and the like, and then the detection is carried out, only mycobacterium tuberculosis has an amplification curve in a specificity test experiment, and other common lung pathogenic bacteria have no amplification curves. The kit provided by the invention has the advantages of rapidness, high sensitivity, simplicity in operation, low cost and the like, and is suitable for popularization and application in detection sites and basic medical institutions.
Drawings
FIG. 1 is an amplification curve of a specificity test performed on a kit provided by the present invention;
FIG. 2 is the results of a sensitivity test performed on the kit provided by the present invention;
FIG. 3 is an amplification curve of a clinical sample experiment performed on the kit provided by the present invention.
Detailed Description
The technical solutions of the present invention are further described in detail with reference to the drawings and the specific embodiments, which are only used for explaining the present invention and are not used for limiting the scope of the present invention.
A kit for rapidly and specifically detecting mycobacterium tuberculosis comprises a working standard substance, a constant-temperature amplification reaction solution and a negative reference substance, wherein the working standard substance comprises a positive reference plasmid of a specific conserved sequence of the mycobacterium tuberculosis, and the specific conserved sequence is SEQ ID No. 1; the isothermal amplification reaction solution comprises 3 pairs of primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; a loop primer LF with a sequence of SEQ ID No. 7; the negative control substance is sputum DNA extracting solution of normal people.
1. The preparation method of the working standard product comprises the following specific steps:
(1) a300 bp long conserved sequence of Mycobacterium tuberculosis (SEQ ID No.1) is synthesized by a chemical synthesis method.
(2) And cloning the synthesized fragment into a pGEM-T vector to construct a positive control plasmid of the mycobacterium tuberculosis.
(3) The obtained positive control plasmid was transformed into E.coli DH 5. alpha. and mass-cultured in LB medium containing 1% ampicillin at 37 ℃ and then extracted with a high purity plasmid Large-Scale kit (DP116) from Tiangen Biochemical technology, Inc. (Beijing).
(4) Plasmid was purified and quantified, and diluted to 104copies/mL, namely the working standardAnd (5) preparing the product.
2. The isothermal amplification reaction solution specifically consists of:
the solvent is double distilled water, and the solute therein is: 8U/20. mu.L Bst DNA polymerase (purchased from NEW ENGLAND BioLabs, cat # M0275), 8mM MgSO4、2.4mM each dNTPs、40mM Tris-HCl、20mM KCl、20mM(NH4)2SO40.2% TritonX-100, 5 pmol/20. mu.L of outer primer F3; 5 pmol/20. mu.L of outer primer B3; 40pmol of inner primer FIP; 40pmol of inner primer BIP; 20pmol of loop primer LB; 20pmol of loop primer LB; 1.25 × EvaGreen fluorescent dye (Biotium, cat # 31000).
3. The negative control substance is DNA extract of normal human sputum, and the extraction method comprises the following steps:
A. 50 μ L of normal human sputum was placed in a sterile centrifuge tube.
Performing thermal denaturation at C.80 deg.C for 15 min, centrifuging at low speed, and collecting supernatant in a new sterilized centrifuge tube to obtain DNA extractive solution of normal human sputum (for experiment or freezing at-20 deg.C).
4. Preparation of DNA specimen of sputum to be detected
The sputum of a possible mycobacterium tuberculosis carrier is taken as a sample for extraction, and the preparation method of the sample to be detected is the same as the preparation method of the sputum DNA extracting solution of the normal person.
5. Reaction system preparation and amplification reaction
At least three reaction tubes: adding 20 mu L of constant-temperature amplification reaction liquid and 5 mu L of sputum DNA specimen to be detected into the first reaction tube; adding 20 mu L of constant temperature amplification reaction liquid and 5 mu L of working standard substance into a second reaction tube; mu.L of isothermal amplification reaction solution and 5. mu.L of negative control were added to the third reaction tube.
And (3) amplification reaction: and (3) reacting the reaction tube for 60min at 65 ℃, testing the fluorescence value in real time, judging whether the sputum DNA sample to be detected carries the mycobacterium tuberculosis according to the amplification curve, and judging that the sputum DNA sample to be detected carries the mycobacterium tuberculosis if the sputum DNA sample to be detected has the amplification curve.
6. Detection of
A. Specificity test
The kit is used for detecting common lung pathogenic bacteria pseudomonas aeruginosa, streptococcus pneumoniae, haemophilus influenzae, legionella pneumophila, mycoplasma pneumoniae, klebsiella pneumoniae, staphylococcus aureus and the like after extracting DNA. The result is shown in fig. 1, only mycobacterium tuberculosis has an amplification curve in a specificity test experiment, and other common pathogenic pulmonary bacteria have no amplification curve, which indicates that the kit provided by the invention has better specificity.
B. Sensitivity test
To a concentration of 104The copies/μ l Mycobacterium tuberculosis standard (i.e. working standard) are respectively 103、102And 10 copies/mu l gradient dilution, and then respectively used as sample DNA to be detected, and the kit is applied to detect the sample DNA. As shown in FIG. 2, it can be seen that the kit can detect about 10 copies/. mu.l at the minimum, and has high sensitivity.
C. Detection of clinical suspected mycobacterium tuberculosis strain
6 sputum containing clinical cases of mycobacterium tuberculosis which are identified by Beijing chest hospitals are collected, DNA is extracted according to the method for preparing the DNA specimen of the sputum to be detected, and then the kit is used for detection. The results are shown in FIG. 3, and 6 clinical cases of Mycobacterium tuberculosis were tested, 6 of which were positive, and the overall positive detection rate was 100%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Flora (Beijing) Biotechnology Ltd
<120> kit for rapidly and specifically detecting mycobacterium tuberculosis and use method thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 300
<212> DNA
<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)
<400> 1
ccgatcgccc catcgaccta ctacgaccac atcaaccggg agcccagccg ccgcgagctg 60
cgcgatggcg aactcaagga gcacatcagc cgcgtccacg ccgccaacta cggtgtttac 120
ggtgcccgca aagtgtggct aaccctgaac cgtgagggca tcgaggtggc cagatgcacc 180
gtcgaacggc tgatgaccaa actcggcctg tccgggacca cccgcggcaa agcccgcagg 240
accacgatcg ctgatccggc cacagcccgt cccgccgatc tcgtccagcg ccgcttcgga 300
<210> 2
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcaaccggga gcccag 16
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<211> 16
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<213> Artificial Sequence (Artificial Sequence)
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tttgccgcgg gtggtc 16
<210> 4
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgtaaacacc gtagttggcg gcttttgcgc gatggcgaac tca 43
<210> 5
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gtgcccgcaa agtgtggcta acttttagtt tggtcatcag ccgttc 46
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acgcggctga tgtgct 16
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<213> Artificial Sequence (Artificial Sequence)
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aggtggccag atgcacc 17
Claims (2)
1. A kit for rapidly and specifically detecting mycobacterium tuberculosis is characterized by comprising a working standard substance, a constant-temperature amplification reaction solution and a negative reference substance,
the working standard substance is a positive control plasmid comprising a specific conserved sequence of mycobacterium tuberculosis, and the specific conserved sequence is SEQ ID No. 1; the positive control plasmid is constructed by cloning a synthetic fragment corresponding to a specific conserved sequence into a pGEM-T vector, and the concentration of the synthetic fragment is 104copies/μL；
The isothermal amplification reaction solution comprises 3 pairs of primers: an outer primer F3, the sequence of which is SEQ ID No. 2; an outer primer B3, the sequence of which is SEQ ID No. 3; the sequence of the inner primer FIP is SEQ ID No. 4; the sequence of the inner primer BIP is SEQ ID No. 5; the sequence of the loop primer LB is SEQ ID No. 6; a loop primer LF with a sequence of SEQ ID No. 7;
the negative control substance is sputum DNA extracting solution of normal people.
2. The kit for rapidly and specifically detecting mycobacterium tuberculosis according to claim 1, wherein the solvent of the isothermal amplification reaction solution is double distilled water, and the isothermal amplification reaction solution contains solutes with the following concentrations: bst DNA polymerase 8U/20. mu.L; 8mM MgSO4；2.4mM each dNTPs；20mM KCl；20mM(NH4)2SO4(ii) a 40Mm Tris-HCl; 0.2 wt% TritonX-100; 5 pmol/20. mu.L of outer primer F3; 5 pmol/20. mu.L of outer primer B3; 40pmol of inner primer FIP; 40pmol of inner primer BIP; 20pmol of loop primer LB; 20pmol of loop primer LB; 1.25 × EvaGreen fluorescent dye.
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